Bacillus pseudomycoides BP101 and application thereof in prevention and treatment of tobacco root rot

Abstract

The invention provides bacillus pseudomycoides BP101 and application thereof, the bacillus pseudomycoides BP101 is named as bacillus pseudomycoides in taxonomy, and the preservation number of the bacillus pseudomycoides BP101 is CCTCC (China Center for Type Culture Collection) NO.20241797. The bacillus pseudomycoides has a good disease prevention effect on tobacco root rot, and has a remarkable growth promoting effect on tobacco plants. The strain has good theoretical research value and application prospect, can be used as a good test material for researching interaction and action mechanism of soil bacteria and plants, can also be used for research and development of soil bio-fertilizers and biological bactericides, can also become a good carrier of multifunctional engineering bacteria for disease prevention, growth promotion and the like, and has broad application prospects. And a new strain resource is provided for biological prevention and control of tobacco root rot.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly relates to a Bacillus pseudomycoides BP101 and its application in preventing and controlling tobacco root rot disease. Background Art

[0002] As an important cash crop in China, tobacco makes a significant contribution to the country's tax revenue and local economy. China has a vast tobacco planting area, and its cigarette production ranks first in the world, making a huge contribution to the national economy. However, in recent years, tobacco root rot disease has spread widely in tobacco planting areas in China and has become an important disease affecting tobacco production. The outbreak of root rot disease not only seriously damages tobacco yield but also has an adverse impact on tobacco quality, bringing huge economic pressure to growers. To address this challenge, the tobacco industry urgently needs to strengthen prevention and control measures to ensure the sustainable development of the industry.

[0003] Tobacco root rot disease is caused by Fusarium spp. and is a common fungal disease affecting tobacco production in China. It can occur both in the seedbed stage and the field stage, and the incidence rate is stable at 3% - 5%. Due to the extension of continuous cropping time and the changes in soil ecological environment and cultivation patterns, Fusarium root rot disease has gradually become the main disease in tobacco production. Currently, the main means of preventing and controlling root rot disease are the use of disease-resistant varieties and chemical control. However, the resources of disease-resistant varieties are limited and the cultivation cycle is long, and chemical control is prone to causing environmental pollution. In contrast, biological control has become a research hotspot due to its characteristics of no residue and environmental friendliness. In particular, actinomycetes in the rhizosphere soil of plants, as natural disease-preventing resources, can show great potential in pest and disease control, promoting plant growth, and enhancing plant disease resistance.

[0004] Therefore, it is necessary to screen and identify a strain from soil or plant materials that can prevent and control root rot disease. Summary of the Invention

[0005] The object of the present invention is to provide a Bacillus pseudomycoides BP101 and its application. This Bacillus pseudomycoides has a good disease-preventing effect on tobacco root rot disease and has a significant growth-promoting effect on tobacco plants.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a Bacillus pseudomycoides BP101 is provided, and the preservation number of the Bacillus pseudomycoides BP101 is: CCTCC NO.20241797.

[0008] In the second aspect of the present invention, a fermentation inoculant is provided, and the fermentation inoculant includes:

[0009] A fermentation broth or a bacterial suspension obtained by fermenting and culturing the Bacillus pseudomycoides BP101;

[0010] Or a dry powder bacterial agent obtained by spray-drying the fermentation broth.

[0011] Furthermore, the preparation method of the bacterial suspension includes:

[0012] A fermentation broth obtained by inoculating the Bacillus pseudomycoides BP101 into a liquid medium for fermentation and culture.

[0013] Furthermore, the conditions for the fermentation culture include: the temperature is 36 - 38 °C, and the pH is 5 - 9.

[0014] In the third aspect of the present invention, there is provided the application of the Bacillus pseudomycoides BP101 or the fermentation inoculant in preventing and controlling tobacco root rot.

[0015] In the third aspect of the present invention, there is provided the application of the Bacillus pseudomycoides BP101 or the fermentation inoculant in promoting the germination of tobacco seeds.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0017] The Bacillus pseudomycoides provided in the embodiments of the present invention has a good disease prevention effect on tobacco root rot and a significant growth-promoting effect on tobacco plants. Thus, it can be seen that this strain has good theoretical research value and application prospects. It can not only be used as a good experimental material for studying the interaction between soil bacteria and plants and its mechanism of action, but also be used for the research and development of soil biological fertilizers and biological fungicides. At the same time, it may also become a good carrier for constructing multi-functional engineering bacteria such as disease prevention and growth promotion, providing a new strain resource for the biological control of tobacco root rot.

[0018] The preservation date of the Bacillus pseudomycoides BP101 of the present invention is August 16, 2024, and the preservation number is CCTCC NO.20241797. Its taxonomic name is Bacillus pseudomycoides BP101, the name of the preservation unit is China Center for Type Culture Collection, the address is Wuhan University, Wuhan, Hubei Province, China, and the postal code is 430072. Description of the Drawings

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0020] Figure 1 It is the bacteriostatic effect diagram of Bacillus pseudomycoides BP101 against Fusarium oxysporum;

[0021] Figure 2 It is the bacteriostatic effect diagram of the sterile fermentation broth;

[0022] Figure 3 It is the disease prevention effect of the strain on tobacco root rot; where Figure A shows the growth of tobacco plant seedlings, and Figure B shows the comparison diagram of the seedlings and the root (rhizome) part;

[0023] Figure 4 It is the strain phylogenetic tree. Detailed implementation manners

[0024] The following will specifically describe the present invention in combination with the detailed implementation manners and embodiments, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and embodiments are used to illustrate the present invention, rather than to limit the present invention.

[0025] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0026] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or by existing methods.

[0027] The technical solution of the embodiment of the present application is to solve the above technical problems, and the general idea is as follows:

[0028] The inventors of the present application isolated a strain of bacteria from tobacco-growing soils collected from disease-free fields in Nanzhang, Zaoyang and other places. Through isolation, purification and screening, it was found that this strain has an antagonistic effect on Fusarium oxysporum f. sp. nicotianae. After colony morphology, biochemical and 16S rRNA sequencing analysis, the homology of this strain with multiple strains of Bacillus pseudomycoides is more than 96%. Combining physiological and biochemical characteristics, this strain was preliminarily determined to belong to Bacillus pseudomycoides and was named Bacillus pseudomycoides.

[0029] The Bacillus pseudomycoides of the present invention has a good promoting effect on tobacco seed germination and tobacco plant growth, and has a good disease prevention effect on tobacco root rot, providing a theoretical basis for the subsequent development of biological bacterial agents.

[0030] The following will combine examples and experimental data to elaborate in detail on a strain of Bacillus pseudomycoides BP101 of the present application and its application in preventing and treating tobacco root rot.

[0031] Example 1: Isolation, purification and identification of Bacillus pseudomycoides BP101

[0032] 1. Isolation and purification of tobacco field soil microorganisms

[0033] Test medium: NA medium: 3 g of beef extract, 1 g of yeast extract, 5 g of peptone, 10 g of glucose, 15 g of agar, made up to 1000 mL with distilled water, pH 7.2, sterilized.

[0034] LB solid medium: 10 g of sodium chloride, 10 g of peptone, 5 g of yeast powder, 1000 mL of water, 15 g of agar, sterilized. Potato dextrose agar (PDA): 200 g of potato, 20 g of glucose, 15 - 20 g of agar, 1000 mL of distilled water, pH 7.0 - 7.4, sterilized.

[0035] Weigh 10 g of soil and place it in a 250 mL conical flask. Then add 100 mL of sterile water, and place the conical flask on an oscillator at 180 r / min for 20 min. Absorb the original solution and prepare a rhizosphere soil suspension with a dilution of 10 -1 ~10 -5 ; Absorb 100 μL of soil suspension with different dilutions of 10 -2 ~10 -5 and spread them evenly on the culture medium plates. Set 2 - 3 parallels for each dilution, and then place them in an incubator at 28 °C for 48 h. After the colonies grow out, conduct 2 - 3 times of streak purification according to various characteristics such as color, morphology, structure, glossiness, etc. Write down the numbers of the purified strains and store them in a refrigerator at 4 °C for later use.

[0036] 2. Isolation and screening of strains

[0037] (1) Primary screening: Using Fusarium oxysporum causing tobacco root rot as the indicator bacterium, the inhibitory activity of the tested strains against Fusarium oxysporum was determined by the plate confrontation method. Activate Fusarium oxysporum on the PAD plate in advance, use a sterile punch with a diameter of 5 mm to punch out a bacterial cake at the edge of the colony, inoculate it in the center of a new PDA plate, and inoculate different bacteria obtained by isolation and purification by parallel streaking 2.5 cm above and below the center of the plate. Using only inoculating the pathogenic bacterium as the blank control. Set 3 replicates for each treatment, and place the plate in an incubator at 28 °C for cultivation. When the control colony grows to 2 / 3 of the culture dish, observe whether there is an obvious antagonistic effect, and screen out the strains with inhibitory effect on the growth of Fusarium oxysporum for the secondary screening.

[0038] 136 strains of bacteria were isolated from the soil. After primary screening, 20 strains with antagonistic effects against tobacco Fusarium were screened out.

[0039] (2) Secondary screening: The 20 strains obtained from the primary screening were subjected to secondary screening by the plate confrontation method. The screening method is the same as above, but after inoculating the fungus on each plate, inoculate the same strain on both sides. The control is not inoculated, and the inhibition zone of the strain is recorded after the plate is fully grown. Take out the above-mentioned primary screening plates, measure the size of the inhibition zone and calculate the inhibition rate. The calculation formula is as follows:

[0040]

[0041] After secondary screening, finally a strain with good antagonistic effect against tobacco Fusarium was screened out, named BP101, and the antagonistic effect is as Figure 1 shown. The antibacterial test results of BP101 of the present invention against tobacco root rot are shown in Table 1.

[0042] Table 1 Antibacterial test of BP101 against tobacco Fusarium

[0043]

[0044] It can be seen from Table 1 that the width of the inhibition zone of strain BP101 against tobacco Fusarium is 18.67 mm, the inhibition rate can reach 62.5%, and the generation of antagonistic effect can be clearly observed on the plate. Therefore, it is selected as the target strain for screening.

[0045] 3. Identification of strains

[0046] Single colonies of the strain were picked and added to LB liquid medium for overnight culture, followed by precipitation and centrifugation. Genomic DNA of the bacteria was extracted, and the gene DNA of the strain was extracted by the boiling water bath method of the bacterial suspension. The bacterial universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1541R (5’-AAGGAGGTGATCCAGCCGCA-3’) were used for PCR amplification. The PCR reaction system included 1 μL of DNA template, 1 μL of each upstream and downstream primer, 12.5 μL of Taq polymerase, and ddH2O was added to make up to 25 μL. The PCR amplification program was: pre-denaturation at 98°C for 5 min, denaturation at 98°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min 30 s, for 35 cycles, and finally extension at 72°C for 5 min. The PCR reaction products were detected by 1% agarose gel electrophoresis. After the PCR amplification products of 16S rRNA were recovered and purified, they were sequenced by Wuhan Qingke Biotechnology Co., Ltd. The sequencing results were submitted to the NCBI database for BLAST homology analysis, and then sequence alignment and phylogenetic tree construction were carried out using MEGA 11.0 software. Data processing was analyzed using Excel and SPSS27 software.

[0047] The 16S rRNA sequence of the test strain obtained by PCR was subjected to BLAST alignment analysis in NCBI. It was found that the sequence homology of this strain with Bacillus pseudomycoides was 99%. Combining the morphological, physiological and biochemical characteristics of this strain, the strain was identified as Bacillus pseudomycoides BP101. This strain has been deposited in the China General Microbiological Culture Collection Center, and the deposit number is CCTCC NO.20241797.

[0048] Example 2: Determination of the antibacterial activity of the sterile fermentation filtrate of the strain

[0049] 1. Preparation of the strain fermentation broth

[0050] The Bacillus pseudomycoides BP101 strain was inoculated into 5 mL of LB liquid medium and cultured with shaking at 37°C and 220 r / min for 12 h to prepare a seed solution. Then, it was inoculated into LB culture medium at an inoculation amount of 1% and cultured under the conditions of 37°C and 220 r / min for 48 h to obtain the strain fermentation broth. Before the pot experiment, the viable bacteria concentration in the fermentation broth was adjusted to 1×10 8 cfu / mL with LB culture medium.

[0051] 2. Determination of the antibacterial activity of the sterile fermentation filtrate of the strain

[0052] (1) Preparation of sterile fermentation filtrate of biocontrol strain: The cultured bacterial liquid fermentation broth was centrifuged at 12,000 r / min for 10 min, and the supernatant was filtered twice through a bacterial filter (0.22 μm) to obtain a sterile fermentation filtrate.

[0053] (2) Effect of sterile fermentation filtrate on the mycelial growth of pathogenic bacteria: The antibacterial effect of the sterile fermentation filtrate of the antagonistic bacterium was tested by the plate punching method. Three holes were evenly punched at a distance of 2.5 cm from the center of the plate (a petri dish with a diameter of 9 mm) using a hole puncher. 40 μL of the fermentation filtrate was injected into each hole, and the sterile LB liquid medium was used as a control. A pathogenic bacteria block with a diameter of 5 mm was inoculated at the center of the medium. Each treatment was repeated 3 times. After culturing in the dark at 28 °C for 5 d, the colony radius of the control group and the treatment group was measured, and the relative antibacterial rate was calculated.

[0054] 3. Results

[0055] The results of the determination of the effect of the sterile fermentation filtrate of the strain on the mycelial growth of the tobacco root rot pathogen are as Figure 2 shown in and Table 2.

[0056] Table 2 Antibacterial activity of the sterile fermentation filtrate of BP101

[0057]

[0058] From Figure 2 the results of and Table 2, it can be seen that the fermentation filtrate of BP101 also has an inhibitory effect on the mycelial growth of Fusarium oxysporum f. sp. nicotianae ( Figure 2 ), the size of the antibacterial zone is 9.67 mm, and the relative antibacterial rate is 30.21%. The antibacterial effect is lower than that of the live bacteria, with a decrease of 46.46% (Table 2).

[0059] Example 3. Determination of the growth promotion effect on tobacco seed germination

[0060] Tobacco seeds with regular and equal particles were selected, surface sterilized with 75% ethanol for 30 s, washed three times with sterile water, and blotted dry with sterile filter paper. Then the seeds were immersed in the bacterial fermentation broth for 4 h. The seeds were taken out and evenly placed at equal distances in a petri dish lined with two layers of moist filter paper, with 50 seeds in each dish. Sterile water (CK1) and LB (CK2) liquid media were used as negative controls, and each treatment was set with 3 replicates. They were cultured in the dark in an illumination incubator for 15 d (humidity 80%, temperature 28 ± 2 °C), and a fixed amount of sterile water was uniformly supplemented every 2 d to keep the filter paper moist. The number of germinated seeds was counted and the germination rate was calculated.

[0061] Seed germination rate = (number of germinated seeds ÷ number of tested seeds) × 100%

[0062] Germination potential (%) = number of seeds germinated in the first 5 days / total number of tested seeds × 100%;

[0063] Germination index (GI) = ∑(Gt / Dt). In the above expression, Gt is the total number of germinated seeds on the t-th day, and Dt is the corresponding germination date;

[0064] Vigor index = germination index × S. In the above expression, S is the total length of the roots and shoots of the seedlings.

[0065] The results are shown in Table 3.

[0066] Table 3 Effects of BP101 on the germination of tobacco seeds

[0067]

[0068] As can be seen from the results in Table 3, 10 days after the seeds were soaked with the strain, the germination rate of the seeds increased significantly, reaching 72.67%, which was 43.34% higher than that of the control group in general, indicating that it has a good promoting effect on the germination of tobacco seeds and the growth of tobacco plants.

[0069] Example 4: Indoor potted plant control efficacy experiment of the strain against tobacco root rot

[0070] The experimental design included a blank group CK1, a control group T1, and a treatment group T2. First, tobacco seedlings with consistent growth were selected. The ends of the roots of the seedlings were cut off by 1 cm using sterilized scissors, 5 roots were cut from each seedling, and the rhizomes were pricked with sterilized needles, with 5 wounds pricked on each rhizome. Then they were transplanted into sterilized nutrient pots (containing nutrient substrates), with 5 plants in each treatment, 3 replicates were set, a total of 15 plants. 2 days after transplantation, the plants were watered by the root irrigation method. Each plant in the blank group and the control group was watered with 15 mL of sterile water; the treatment group was watered with the biocontrol bacteria fermentation broth prepared in Example 2 according to the amount of 15 mL per plant. 48 hours later, the control group and the treatment group were watered with a Fusarium oxysporum suspension according to the amount of 15 mL per plant. Among them, the concentration of fungal spores in the suspension was 5×10 6 per mL, and the concentration of the biocontrol bacteria fermentation broth was 1×10 5 CFU / mL. The disease incidence was counted after the control group became ill after growing for 15 days. The grading standard for tobacco root rot refers to the Tobacco Industry Standard of the People's Republic of China (GB / T 23222-2008).

[0071] Disease incidence = (number of diseased plants / total number of plants surveyed) × 100%

[0072] Disease index = [∑(number of diseased plants or leaves at each level × the value of that disease level) / (total number of plants or leaves surveyed × the highest disease level value)] × 100

[0073] Relative control efficacy = [(control disease index - treatment disease index) / control disease index] × 100%

[0074] The results are as Figure 3As shown in A, the tobacco seedlings in the control group showed wilting and yellowing. Separately, from Figure 3 As can be seen from B, the roots and stems of the seedlings in the control group inoculated with the pathogenic bacteria showed rot, the plants died, and pathological phenotypes similar to those in the field were produced. However, the seedlings in the treatment group (inoculated with BP101) showed no disease symptoms, and the roots and stems grew normally without rot symptoms, proving that inoculating pathogenic bacteria indoors could cause tobacco to develop and die, but inoculating the potential biocontrol bacterium BP101 could effectively prevent the plants from developing and dying. The specific disease statistics are as follows:

[0075] Table 4 Inhibitory effect of biocontrol bacterium HJB-XTBG45 on the incidence of tobacco root rot

[0076]

[0077] As can be seen from Table 4, among the control group, 15 tobacco seedlings showed wilting, yellowing and death caused by Fusarium oxysporum, and the incidence of root rot reached 93.33%. While the incidence of tobacco seedlings in the treatment group inoculated with BP101 was 13.33%. At the same time, the disease index of the diseased plants was reduced, and the relative control effect was 79.94%.

[0078] In summary, a Bacillus pseudomycoides BP101 provided by the present invention has a good disease prevention effect on tobacco root rot and a significant growth promotion effect on tobacco plants.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

[0080] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0081] Although the embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the embodiments and all changes and modifications falling within the scope of the present invention.

[0082] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A Bacillus pseudomycoides BP101, characterized in that, The taxonomic nomenclature of the Bacillus pseudomycoides is Bacillus pseudomycoides BP101, and its deposit number is: CCTCC NO. 20241797.

2. A fermentation inoculant, characterized in that, The fermentation inoculum includes: The fermentation broth or bacterial suspension obtained by fermenting and culturing the Bacillus pseudomycoides BP101 as described in claim 1; Or the dry powder inoculum obtained by spray-drying the fermentation broth.

3. The fermenting inoculant according to claim 2, characterized in that, The preparation method of the bacterial suspension includes: The fermentation broth obtained by inoculating the Bacillus pseudomycoides BP101 as described in claim 1 into a liquid medium for fermentation and culture.

4. The fermenting inoculant according to claim 3, characterized in that, The conditions for the fermentation culture include: the temperature is 36 - 38 °C, and the pH is 5 - 9.

5. Use of the Bacillus pseudomycoides BP101 as described in claim 1 or the fermentation inoculum as described in any one of claims 2 - 4 in preventing and controlling tobacco root rot.

6. Use of the Bacillus pseudomycoides BP101 as described in claim 1 or the fermentation inoculum as described in any one of claims 2 - 4 in promoting tobacco seed germination and tobacco plant growth.

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